In 1964, Levin and Bang discovered the phenomenon in which horseshoe crab (limulus) amebocyte lysate (hereinafter sometimes abbreviated as LAL) is immediately coagulated (gelled) by a Gram-negative bacterial endotoxin [J. Levin and F. B. Bang: Bull. Johns Hopkins Hospital, 115, 265-274 (1964)]. Since then, LAL has been widely utilized as the so-called Limulus Test reagent in a method of specific detection of endotoxins. Today, three genera, four species of horseshoe crab survive throughout the world and Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda are known. "Limulus Test" reagents comprising the amebocyte lysate of L. Polyphemus occurring in the United States and T. tridentatus occurring in Japan and China have been commercialized. [See, for example, Progress in Clinical and Biological Research: volume 93; "Endotoxins and their Detection with the Limulus Amebocyte Lysate Test", edited by Stanly W. Watson, Jack Levin and Thomas J. Novitsky, published in 1982 by Alan R. Liss Inc., pages 7-24, entitled: The Limulus Test and Bacterial Endotoxins: Some Perspectives by J. Levin.]
LAL was first considered to react specifically only with endotoxins. Recent studies have shown that LAL has been found to react with (1.fwdarw.3)-.beta.-D-glucan as well as endotoxin. The coagulation system of LAL, like the mammalian blood coagulation system, consists of two or more cascade reactions of coagulation factors, and include not only an endotoxin-mediated pathway (factor C pathway), but also a pathway to be triggered by (1.fwdarw.3)-.beta.-D-glucan (factor G pathway) [T. Morita et al., FEBS LETTERS, 129, 318-321 (1981), and S. Iwanaga et al., J. Protein Chem., 5, 255-268 (1986)]. Accordingly, work has been done in order to render the Limulus test as endotoxin-specific as possible. For example, T. Obayashi et al., Clin. Chim. Acta, 149, 55-65 (1985) proposed a method of determining an endotoxin by using a reagent obtained by removing factor G from LAL by separation and reconstitution of the coagulation factors. An endotoxin specific assay kit in accordance with this method is sold under the tradename "Endospecy.RTM." by Seikagaku Kogyo Co., Ltd.
The above-proposed assay method has a very strong demand as an edndotoxin-specific assay. However, this method involves certain disadvantages to be described.
(1) To separate and remove factor G from limulus amebocyte lysate composed of a plurality of coagulation factors, it is necessary to perform the operation of separating the individual factors in the absence of an endotoxin or (1.fwdarw.3)-.beta.-D-glucan. Accordingly, the endotoxin or (1.fwdarw.3)-.beta.-D-glucan must be removed completely in advance from tools, devices and chemicals used in the separating operation for fractionation.
(2) As the separating operation proceeds, the amebocyte lysate becomes diluted, and it must occasionally be concentrated.
(3) Every time a separating operation is carried out, the factors decrease in activity or a loss of the fractions occurs.
(4) The coagulogen (clottable protein precursor) is separated and removed together with factor G.
Because of these disadvantages, the above proposed assay method can be applied only to a chromogenic method, and cannot be applied to methods utilizing the gellation phenomenon such as a gellation method, turbidimetry and turbidimetric kinetic assay. In the course of studying a pathway to be triggered by (1.fwdarw.3)-.beta.-D-glucan (factor G pathway) in the LAL coagulation mechanism, the present inventors unexpectedly found that among (1.fwdarw.3)-.beta.-D-glucans heretofore considered to be involved only in the activation of factor G, one containing a structural portion consisting of a specific number of continuously bound (1.fwdarw.3)-.beta.-D-glucoside structural units, quite contrary, shows a factor G inhibiting action. This finding has led to accomplishment of the present invention.